Vaccinia virus polypeptides

ABSTRACT

This document provides methods and materials related to polypeptides present in a vaccinia virus (e.g., polypeptides that can be isolated from naturally processed and presented class I polypeptides originating from vaccinia virus, a member of the Orthopoxvirus family). For example, methods for generating a vaccine comprising one or more of vaccinia virus polypeptides disclosed herein for preventing or treating Orthopoxvirus infection are provided. In addition, kits related to the use of vaccinia polypeptides are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 13/222,862, filed Aug. 31, 2011, which claims the benefit of U.S. Provisional Application Ser. No. 61/379,311, filed Sep. 1, 2010. The disclosure of the prior applications are considered part of (and are incorporated by reference in) the disclosure of this application.

BACKGROUND

1. Technical Field

This document provides methods and materials relating to isolated vaccinia virus-derived polypeptides. For example, this document relates to specific and naturally processed and HLA presented vaccinia virus-derived polypeptides isolated from vaccinia virus, a member of the Orthopoxvirus family. This document provides methods for generating a vaccine for preventing or treating Orthopoxvirus infection that induces a protective therapeutic immune response. The vaccines can include one or more of the isolated vaccinia virus polypeptides provided herein. In addition, this document provides kits related to the use of vaccinia virus polypeptides.

2. Background Information

Polypeptide-based vaccines use small polypeptide sequences derived from target proteins as epitopes to provoke an immune reaction. These vaccines are a result of an improved understanding of the molecular basis of epitope recognition, thereby permitting the development of rationally designed, epitope-specific vaccines based on motifs demonstrated to bind to human class I (HLA I) or class II (HLA II) major histocompatibility complex (MHC) molecules. Of particular interest has been the discovery of epitopes that are specifically recognized by T cells for prophylaxis and treatment of infectious diseases.

Over the centuries, naturally occurring smallpox, with its case-fatality rate of 30 percent or more and its ability to spread in any climate and season, has been universally feared as one of the most devastating of all the infectious diseases. The use of vaccinia virus as a vaccine enabled the global eradication of naturally occurring smallpox. The last naturally occurring case of smallpox occurred in Somalia in 1977. In May 1980, the World Health Assembly certified that the world was free of naturally occurring smallpox. Routine vaccination against smallpox in the United States ended in 1971, and except for some soldiers and laboratory workers, no one has been vaccinated since 1983. However, terrorist activities in the early 21st century as well as imported outbreaks of monkeypox (a member of the Orthopox virus family) in the USA, spurred renewed interest in biodefense countermeasures for these public health threats (Artenstein et al., Expert Rev. Vaccines, 7:1225-1237 (2008) and Giulio et al., Lancet Infect. Dis., 4:15-25 (2004)).

SUMMARY

This document provides methods and materials related to vaccinia virus polypeptides. For example, this document provides vaccinia virus polypeptides that have the ability to be naturally processed and presented by HLA molecules. This document also provides methods and materials (e.g., vaccines) for preventing or treating Orthopoxvirus infections. For example, the vaccines provided herein can include one or more of the vaccinia virus polypeptides provided herein and can have the ability to induce a protective therapeutic immune response within a mammal (e.g., a human). In addition, this document provides kits related to the use of vaccinia virus polypeptides.

As described herein, two-dimensional liquid chromatography coupled to mass spectrometry was used to identify 116 vaccinia virus polypeptides, encoded by 61 open reading frames, from a human B-cell line (homozygous for HLA class I A*0201, B*1501, and C*03) after infection with vaccinia virus (Dryvax). The identification of these naturally processed and presented polypeptides resulting from vaccinia virus infection can be used to aid in understanding the immune process and can be used to generate vaccines against Orthopoxviruses.

In general, one aspect of this document features an isolated polypeptide, wherein the amino acid sequence of the polypeptide is as set forth in any one of SEQ ID NOs:1-83.

In another aspect, this document features a composition comprising at least one isolated polypeptide selected from the group consisting of SEQ ID NOs:1-82 and 83 and at least one polypeptide selected from the group consisting of SEQ ID NOs:84-115 and 116. The composition can further comprise an adjuvant.

In another aspect, this document features a method of preventing or treating variola virus infection in a subject. The method comprises, or consists essentially of, administering to the subject a composition comprising an adjuvant and at least one polypeptide, wherein the amino acid sequence of the polypeptide is as set forth in any one of SEQ ID NOs:1-83. The subject can be a human.

In another aspect, this document features a vaccine comprising, or consisting essentially of, at least one isolated polypeptide, wherein the amino acid sequence of the polypeptide is as set forth in any one of SEQ ID NOs:1-83. The vaccine can comprise at least one polypeptide selected from the group consisting of SEQ ID NOs:84-115 and 116. The vaccine can comprise an adjuvant.

In another aspect, this document features a method of enhancing the immune response in a subject to a vaccine. The method comprises, or consists essentially of, administering an agent capable of increasing the expression of a transporter associated with antigen processing in the subject, wherein the vaccine comprises at least one isolated polypeptide, wherein the amino acid sequence of the polypeptide is as set forth in any one of SEQ ID NOs:1-83. The transporter can be TAP1, TAP2, or Tapasin.

In another aspect, this document features a method of inducing an immune response against at least one isolated polypeptide selected from the group consisting of SEQ ID NOs:1-82 and 83. The method comprises, or consists essentially of, administering the polypeptide to a subject in an amount effective to induce an immune response against the polypeptide. The polypeptide can be administered in combination with a polypeptide selected from the group consisting of SEQ ID NOs: 84-115 and 116. The polypeptide can be administered in combination with a pharmaceutically acceptable excipient, carrier, diluent, or vehicle. The method can comprise administering to the subject an agent capable of increasing expression of a TAP molecule. The immune response can be a cell mediated immune response. The cell mediated immune response can be a cell mediated cytolytic immune response. The cell mediated immune response can be a class I-restricted T cell response.

In another aspect, this document features a kit comprising, or consisting essentially of, (a) at least one polypeptide selected from the group consisting of SEQ ID NOs:1-82 and 83, and (b) an adjuvant. The kit can comprise at least two polypeptides selected from the group. The kit can comprise at least one polypeptide selected from the group consisting of SEQ ID NOs:84-115 and 116.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram of a protocol for isolating and identifying HLA class I polypeptides from B cells infected with vaccinia virus.

FIG. 2 contains graphs plotting the distribution of HLA polypeptide amino acid length for (A) vaccinia virus polypeptides and (B) all identified polypeptides, and the putative sorting of polypeptides by binding allele for (C) vaccinia virus polypeptides and (D) all identified polypeptides. Polypeptides were classified by allele using their C-terminal amino acid: L or V assigned to A*0201, F or Y assigned to B*1501, and all other polypeptides marked as NA (not assigned).

FIG. 3A is a graph of the MS/MS spectrum of the vaccinia virus polypeptide IQYPGSEIKGNAY (SEQ ID NO:16) found in SCX fractions 4 and 5, as annotated by Scaffold, including mass accuracy of the precursor mass in parts-per-million (ppm). A table of the fragment ions matched and the experimental error of the fragment ions is included. The Orbitrap survey spectrum of the precursor ion is shown. FIG. 3B is a graph containing the same information for the vaccinia virus polypeptide IQYPGSKIKGNAY (SEQ ID NO:17) as identified from SCX fractions 14-16. Note the different precursor mass, as well as concomitant changes to fragment ion masses consistent with the change in amino acid E (Glu) to K (Lys).

FIG. 4 is a pie graph of vaccinia epitopes directly identified by MS/MS that are classified by predicted HLA-binding strength as determined by the netMHC algorithm at the Center for Biological Sequence Analysis, Technical University of Denmark, (“http” colon, slash, slash “www” dot “cbs.dtu.dk” slash “services” slash “NetMHC” slash). Polypeptide sequences with calculated IC₅₀ values<50 nM were classified as strong binding, IC₅₀ values between 50 nM and 500 nM were classified as weak binding, and IC₅₀>500 nM were classified as non-binding polypeptides.

DETAILED DESCRIPTION

This document provides methods and materials related to vaccinia virus polypeptides. For example, this document provides vaccinia virus polypeptides that have the ability to be naturally processed and presented by HLA molecules. This document also provides methods and materials (e.g., vaccines) for preventing or treating Orthopoxvirus infections. For example, the vaccines provided herein can include one or more of the vaccinia virus polypeptides provided herein and can have the ability to induce a protective therapeutic immune response within a mammal (e.g., a human). In addition, this document provides kits related to the use of vaccinia virus polypeptides.

This document provides compositions (e.g., vaccine compositions) containing one or more vaccinia virus polypeptides provided herein. In some cases, a vaccinia virus polypeptide provided herein can have the ability to be naturally processed and presented by a class I MHC molecule. Examples of such vaccinia virus polypeptide provided herein include, without limitation, those vaccinia virus polypeptides set forth in SEQ ID NOs:1-83 of Table 2. In some cases, the polypeptides set forth in SEQ ID NOs:1-83 can be used individually or as a mixture for the prevention and/or therapeutic treatment of Orthopoxvirus infections in vitro and in vivo, and/or for improved diagnostic detection of Orthopoxvirus infections. Any appropriate combination of the polypeptides listed in Table 2 can be used. For example, the combination can include at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more polypeptides selected from Table 2. For example, the polypeptides corresponding to SEQ ID NOs: 1-10 can be used in any combination. In some cases, the polypeptides corresponding to SEQ ID NOs:1-10 and SEQ ID NOs:70-83 can be used in any combination. For example, the polypeptides corresponding to SEQ ID NO:1 and SEQ ID NO:3 can be used in any combination with SEQ ID NOs:70-83. In some cases, a combination of the polypeptides listed in Table 2 can be used with the exception of 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more polypeptides selected from Table 2. For example, the polypeptides corresponding to SEQ ID NOs:1-10 can be used in any combination with the exception of SEQ ID NOs:11-83. For example, the polypeptides corresponding to SEQ ID NOs:1-83 can be used in any combination with the exception of SEQ ID NO:10, SEQ ID NO:20 and SEQ ID NO:30.

In some cases, one or more of the polypeptides set forth in SEQ ID NOs:1-83 can be used in combination with at least one of the polypeptides set forth in SEQ ID NOs:84-116 of Table 3. Any appropriate combination of the polypeptides listed in Table 2 can be used with at least one of the polypeptides set forth in SEQ ID NOs:84-116 of Table 3. In some cases, a combination can include at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more polypeptides selected from Table 2 with at least one of the polypeptides set forth in Table 3. For example, the polypeptides corresponding to SEQ ID NOs:1-10 from Table 2 can be used in combination with SEQ ID NO:84 of Table 3. In some cases, the polypeptides corresponding to SEQ ID NOs:1-10 and SEQ ID NOs:70-83 can be used in combination with SEQ ID NO:84. In some cases, the combination can include at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more polypeptides selected from Table 2 with at least 2, 3, 5, 10, 15, 20, 25, or more polypeptides selected from Table 3. For example, the polypeptides corresponding to SEQ ID NOs:1-10 can be used in combination with SEQ ID NOs:84-90. In some cases, SEQ ID NOs:1, 5, and 10 can be used in combination with SEQ ID NOs:85, 90, and 100. In some cases, one or more vaccinia virus polypeptides set forth in SEQ ID NOs: 19, 29, and 49 can be used in combination with one or more vaccinia virus polypeptides set forth in SEQ ID NOs:41 and 42.

In some cases, a composition can be designed to include one or more vaccinia virus polypeptides that have a sequence present within a vaccinia virus polypeptide that is expressed during an early phase of a poxvirus infection in combination with one or more vaccinia virus polypeptides that have a sequence present within a vaccinia virus polypeptide that is expressed during a late phase of a poxvirus infection. For example, one or more vaccinia virus polypeptides set forth in Table 2 or Table 3 for ORFs A7L, D13L, D6R, DBL, E10R, E6R, EBR, G4L, H4L, H7R, and I1L (e.g., a polypeptide involved in a late phase of infection) can be used in combination with one or more vaccinia virus polypeptides set forth in Table 2 or Table 3 for ORFs A44L, A46R, A48R, A52R, ABR, B12R, B13R, B15R, B1R, C11R, C12L, C2L, ESR, E9L, F11L, F12L, F16L, F1L, HSR, J3R, J4R, J6R, K1L, K3L, K6L, K7R and N2L (e.g., a polypeptide involved in an early phase of infection). In some cases, one or more vaccinia virus polypeptides set forth in SEQ ID NOs:19 (A44L) and 49 (E5R) can be used in combination with one or more vaccinia virus polypeptides set forth in SEQ ID NOs:29 (A7L), 41 (D13L), and 42 (D13L).

In some cases, a composition can be designed to include two or more vaccinia virus polypeptides (e.g., two, three, four, five, six, seven, eight, nine, ten, or more vaccinia virus polypeptides) that potentially have the ability to bind to class I MHC molecules with high binding affinity. For example, two or more vaccinia virus polypeptides set forth in SEQ ID NOs:1, 19, 29, 37, 41, 42, 44, 49, 64, and 68 can be used in combination to form a composition (e.g., a vaccine composition).

The polypeptides provided herein (e.g., the polypeptide presented in Tables 2 and 3) can include oxidized amino acid residues (e.g., oxidized forms of methionine) or can lack oxidized amino acid residues.

The term “isolated” refers to material which is substantially or essentially free from components which normally accompany the material as it is found in its native state. Thus, isolated polypeptides as described in this document do not contain materials normally associated with the polypeptides in their in situ environment. The term “polypeptide” generally refers to a short chain of amino acids linked by polypeptide bonds. Typically, polypeptides comprise amino acid chains of about 2-100, more typically about 4-50, and most commonly about 6-20 amino acids.

Any appropriate method can be used to obtain a vaccinia virus polypeptide provided herein. For example, polypeptides having the sequence set forth in any one of SEQ ID NOs:1-116 can be synthesized by methods known to one skilled in the art of making polypeptides. Of course, other methods in the art would be appropriate. In some cases, simple chemical polypeptide synthesis techniques can be used to obtain a vaccinia virus polypeptide provided herein. In some cases, a polynucleotide sequence encoding for a vaccinia virus polypeptide of interest can be inserted into a plasmid or other vector that can then be delivered to hosts that can be induced to transcribe the polynucleotide into the polypeptide of interest. In some cases, a polynucleotide sequence for a larger polypeptide can be inserted into host cells that can produce the larger polypeptide and then process that polypeptide into a smaller polypeptide or a functionally equivalent variant of interest.

A composition provided herein containing one or more polypeptides set forth in SEQ ID NOs:1-83 of Table 2 or any appropriate combination of polypeptides as described herein can be formulated to provide a polypeptide-based vaccine. In some cases, such a vaccine can be designed to be based on a combination of naturally processed and presented vaccinia virus polypeptides. For example, a polypeptide-based vaccine can be designed to include at least one polypeptide selected from SEQ ID NOs:1-83 and at least one polypeptide selected from SEQ ID NOs:84-116. Any appropriate method can be used to formulate a polypeptide-based vaccine including, for example, those methods used to formulate polypeptide-based vaccines directed against other viral targets. Examples of polypeptide-based vaccines directed to other viral targets are described elsewhere (see, e.g., Belyakov et al., Proc. Natl. Acad. Sci. U.S.A., 95(4):1709-1714 (1998) and Jackson et al., Proc. Natl. Acad. Sci. U.S.A., 101(43):15440-15445 (2004)). In some cases, a vaccine composition provided herein can include one or more polypeptides set forth in SEQ ID NOs:1-83 (or any appropriate combination of polypeptides as described herein) in combination with the active ingredients or polypeptides of a vaccine composition described elsewhere (see, e.g., Belyakov et al., Proc. Natl. Acad. Sci. U.S.A., 95(4):1709-1714 (1998) and Jackson et al., Proc. Natl. Acad. Sci. U.S.A., 101(43):15440-15445 (2004)). Such vaccine composition can provide a level of protection against Orthopoxvirus infections as well as infections by the other viral targets.

In some cases, a vaccine composition provided herein can be designed to prevent or treat an Orthopoxvirus infection. For example, a vaccine composition provided herein can have the ability to induce a protective or therapeutic immune response within a mammal (e.g., a human). In some cases, a vaccinia virus polypeptide provided herein can be a highly conserved polypeptide across the family of Orthopoxvirus members. In such cases, a vaccine composition containing such a highly conserved polypeptide can be used to provide protection against multiple members of the Orthopoxvirus family. In some cases, a vaccine composition provided herein can be directed against any Orthopoxvirus. For example, a vaccine composition provided herein can be directed against monkeypox, cowpox, and camelpox. In some cases, a vaccine composition provided herein can be directed against vaccinia or variola major or minor. The term “vaccine” as used herein refers to immunogenic compositions that are administered to a subject for the prevention, amelioration, or treatment diseases, typically infectious diseases. In some cases, one or more features of other vaccine preparations can be incorporated into a vaccine composition provided herein. For example, a polypeptide used to create a vaccinia vaccine can be included within a vaccine composition provided herein. Examples of vaccinia-specific single polypeptide vaccines that have one or more features that can be included in the methods and materials (e.g., a vaccine composition) provided herein are described elsewhere (see, e.g., Snyder et al., J. Virol., 78(13):7052-60 (2004) and Drexler et al., Proc. Natl. Acad. Sci. U.S.A., 100(1):217-22 (2003)).

A polypeptide provided herein (e.g., a polypeptide set forth in Table 2 or Table 3) can be formulated into a vaccine composition using any appropriate method.

In some cases, a polypeptide provided herein can be combined with a pharmaceutically acceptable carrier or pharmaceutical excipient. The term “pharmaceutically acceptable” refers to a generally non-toxic, inert, and/or physiologically compatible composition. A term “pharmaceutical excipient” includes materials such as adjuvants, carriers, pH-adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservatives, and the like. Examples of adjuvants include, without limitation, CpG, aluminum sulfate, aluminumphosphylate, and MF59. In some cases, vaccines or components of a vaccine can be conjugated to, for example, a polysaccharide or other molecule, to improve stability or immunogenicity of one or more vaccine components. In some cases, a polypeptide provided herein (e.g., a polypeptide set forth in Table 2 or Table 3) can be formulated into a vaccine composition containing cells. For example, one or more polypeptides provided herein can be included within a cellular vaccine. Any appropriate method can be used to prepare a cellular vaccine or the components of a cellular vaccine.

The methods and materials provided herein can be used in combination with other techniques having the ability to enhance the immune response of a vaccine. For example, a vaccine composition provided herein can be designed to include or to be used in combination with an effective amount of an agent that can augment the level of a TAP molecule and/or a tapasin molecule within a cell. Increasing the level of TAP and/or tapasin molecules within a cell can increase the immunogenicity of a vaccine composition containing a polypeptide provided herein. In some cases, the techniques described elsewhere (Vitalis et al., PLoS Pathog., 1(4):e36 (2005)) can be used in combination with the methods and materials provided herein. Examples of TAP molecules include, without limitation, TAP-1 molecules and TAP-2 molecules. In some cases, an effective amount of an agent that can augment the level of a TAP1 molecule alone, a TAP2 molecule alone, both TAP-1 and TAP-2 molecules, a tapasin molecule alone, or the combination of TAP-1, TAP-2, and tapasin molecules can be used in combination with the methods and materials provided herein.

The levels of a TAP molecule can be augmented using agents that can increase TAP expression including, without limitation, interferon-γ and p53. In some cases, the levels of a TAP molecule or a tapasin molecule can be augmented by administering a nucleic acid molecule encoding a TAP molecule or a tapasin molecule. The target cell can be any appropriate cell to which one wishes to generate an immune response. For example, in a prophylactic therapy or a vaccine, the target cell can be an essentially normal cell (e.g., a cell expressing normal TAP levels) that may not have been otherwise exposed to an antigen. In such a case, the agent that augments TAP can be co-administered with the antigen (e.g., a polypeptide provided herein) to which one wishes to generate an immune response. For example, when used as a therapeutic, the target cell can be previously infected with a pathogen (such as a virus or bacteria).

This document also provides methods and materials for treating mammals (e.g., humans) having an Orthopoxvirus infection. For example, a composition provided herein can be administered to a mammal having an Orthopoxvirus infection under condition effective to reduce the severity of one or more symptoms of the Orthopoxvirus infection. Treatment of individuals having an Orthopoxvirus infection (e.g., a vaccinia virus infection) can include the administration of a therapeutically effective amount of one or more polypeptides set forth in SEQ ID NOs:1-83. In some cases, treatment can include the use of one or more polypeptides set forth in SEQ ID NOs:1-83 individually or as a mixture. In some cases, one or more polypeptides set forth in SEQ ID NOs:1-83 can be used in combination with at least one of the polypeptides set forth in SEQ ID NOs:84-116. The polypeptides can be used or administered as a mixture, for example, in equal amounts, or individually, provided in sequence, or administered all at once. The term “therapeutically effective amount” refers to that amount of the agent sufficient to result in amelioration of symptoms, e.g., treatment, healing, prevention or amelioration of such conditions. In providing a subject with a polypeptide provided herein (e.g., a vaccinia-derived polypeptide), combinations or fragments thereof, capable of inducing a therapeutic effect, the amount of administered agent will vary depending upon such factors as the subject's age, weight, height, sex, general medical condition, previous medical history, etc. In some cases, the amount of administered agent can vary depending upon the subject's HLA allele type. The subject can be, for example, a mammal. The mammal can be any type of mammal including, without limitation, a mouse, rat, dog, cat, horse, sheep, goat, cow, pig, monkey, or human.

This document also provides kits that can be used for a variety of applications including, without limitation, combining reagents necessary for producing vaccine compositions. Such vaccine compositions can include one or more polypeptides provided herein (e.g., one or more vaccinia-derived polypeptide described herein) as well as adjuvants, diluents, and pharmaceutically acceptable carriers. In some cases, a kit provided herein can include a combination of vaccinia virus polypeptides (e.g., vaccinia-derived polypeptides) as described herein. In some cases, a kit provided herein can include at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more polypeptides described herein. Any appropriate combination of the polypeptides can be used. In some cases, a kit provided herein can include one or more adjuvants and can include instructions for preparing and administering a vaccine composition.

In some cases, a kit provided herein can be designed as a diagnostic kit. For example, a kit provided herein can be designed to include reagents that can be used to detect cellular immune responses. In some cases, a kit provided herein can be designed to include polypeptides that can be used to detect antigen specific T cells. Such polypeptides (e.g., a polypeptide listed in Table 2 or 3) can be used to detect antigen specific T cells in samples from Orthopoxvirus (e.g., vaccinia virus) infected or exposed subjects. In some cases, such polypeptides (e.g., a polypeptide listed in Table 2 or 3) can be used to detect antigen specific T cells post-vaccination of a subject to determine the efficacy of immunization.

Any appropriate method can be used to detect antigen specific T cells using a polypeptide provided herein. For example, flow cytometry, enzyme-linked immunospot (ELISPOT), cytokine secretion, direct cytotoxicity assays, and lymphoproliferation assays can be used to detect antigen specific T cells using a polypeptide provided herein. In some cases, flow cytometry using MHC class I tetramers can be used, particularly for vaccinia epitope specific quantitation of CD8⁺ T cells. Such kits can include at least one polypeptide provided herein. In some cases, such a kit can include at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more polypeptides provided herein for the detection of antigen specific T cells.

The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES Example 1 Discovery of Naturally Processed and HLA-Presented Class I Polypeptides from Vaccinia Virus Infection using Mass Spectrometry for Vaccine Development

Materials and Methods

Cell Culturing and Vaccinia Virus Infection

Epstein-Barr virus (EBV)-transformed B-cells (European Collection of Cell Cultures—ECACC, number 86052111, Salisbury, Wiltshire, UK), homozygous for the HLA-A*0201, B*1501, and C*03 were of human origin and used as antigen presenting cells (APCs). The New York City Board of Health (NYCBOH, Dryvax®) vaccine-strain of Vaccinia virus was cultured in HeLa cells in Dulbecco's modified Eagle's medium, supplemented with 5% fetal calf serum (FCS; Life Technologies, Gaithersburg, Md.). B-cells were infected with live Vaccinia virus at a multiplicity of infection (moi) of 0.1 PFU/cell for 2 hours and further maintained for 24-30 hours in RPMI-1640 supplemented with 8% FCS. These uninfected and Vaccinia-infected cells (approximately 1×10⁹ cells each) were used for obtaining cell lysates for the class I HLA molecules purification.

Isolation of HLA-Associated Polypeptides from Uninfected and Vaccinia-Infected Cells

Class I HLA molecules were purified from human homozygous B-cells using an immunoaffinity approach, and their associated polypeptides were extracted as previously described (Slingluff et al., J. Immunol., 150:2955-2963 (1993) and Johnson et al., J. Am. Soc. Mass Spectrom., 16(11):1812-1817 (2005)). In brief, cells were lysed with a buffer containing 20 mM Tris, pH 8.0, 150 mM NaCl, 1% CHAPS and protease inhibitors (1 mM Pefabloc SC, Roche Applied Science, Indianapolis, Ind.). The clarified supernatants were passed over a protein A-sepharose 4B (Sigma) column containing the monoclonal antibody (mAb) W6/32 specific for HLA-A, B and C (Parham et al., J. Immunol., 123(1):342-349 (1979) and Hogan et al., Cancer Immunol. Immunother., 54(4):359-371 (2005)). The HLA molecules (1.2 mg/mL) were dissociated from their bound class I polypeptides using 0.2N acetic acid, pH 2.7, and polypeptides were separated from the HLA by filtration through a 10-kDa molecular mass cutoff filter (Millipore, Bedford, Mass.).

Strong Cation Exchange Fractionation

Strong cation exchange (SCX) fractionation of the sample was performed after desalting the polypeptide pool with a reversed phase 1 mm by 8 mm polypeptide trap (Michrom BioResources, Auburn, Calif.). SCX chromatography used a gradient of 5 mM KH₂PO₄, pH 3.0 to 0.4 M KCl of 5 mM KH₂PO₄, pH 3.0. Acetonitrile was added to each mobile phase to 20% by volume. Desalted polypeptides were loaded onto a polysulfoethyl aspartamide column (Michrom BioResources) at 0.5% mobile phase A, and a gradient was developed to 20% B over 20 minutes at a flow rate of 200 μL/min, then from 20% B to 80% B over the next 10 minutes. Two-minute fractions were collected; each fraction was vacuum centrifuged to dryness before analyzing the fractions by nLC-MS/MS.

LTQ-Orbitrap nLC-MS/MS Analyses

Automated nLC-MS/MS analyses were performed on a commercial linear ion trap-Fourier transform hybrid mass spectrometer (LTQ-Orbitrap, Thermo Fisher Scientific, Waltham, Mass.), interfaced to a nano-scale liquid chromatograph and autosampler (Eksigent NanoLC 1D, Dublin, Calif.), using a 15 cm by 75 μm i.d. column packed with Magic C_(18AQ) (5 μm particles, 200 Å pore size, Michrom BioResources). The autosampler loaded 5-20 μL onto a 0.25 μL pre-column (Optimize Technologies, Oregon City, Oreg.), custom-packed with Magic C₈, 5 μm, 200 Å (Michrom BioResources). Mobile phase A consisted of water/acetonitrile/formic acid (98/2/0.2 by volume) and mobile phase B was acetonitrile/water/formic acid (90/10/0.2 by volume). A 90 minute LC method employed a gradient from 2% to 40% B over 60 minutes, followed by a second segment to 90% B at 85 minutes, with a column flow of 0.4 μL/minute. A third pump was used to load polypeptides from the autosampler to the pre-column, with 0.05% TFA and 0.15% formic acid in water at 15 μL/minute.

SCX fractions were analyzed multiple times by nLC-MS/MS using data-dependent acquisition of tandem mass spectra. The first experiment targeted singly charged precursors between 750 and 1500 on the m/z (molecular mass m divided by charge z) scale. A second experiment targeted doubly and triply charged precursors between m/z 375 and 750, consistent with MHC class I polypeptides which are predominantly 9-11 amino acids long.

The LTQ-Orbitrap was operated in a data-dependent mode, first acquiring an Orbitrap survey scan with 60,000 resolving power (FWHM at m/z 400), a target cell population of 1×10⁶ ions, and a maximum ion fill time of 300 ms. The preview Fourier transform was used to select the five most abundant ions for MS/MS experiments in the LTQ. LTQ MS/MS spectra were acquired with a 2.5 mass unit isolation width, target ion population of 1×10⁴ ions, one microscan, maximum ionization fill time of 100 ms, normalized collision energy of 35%, activation Q of 0.25, and activation time of 30 ms. Once ions were selected for MS/MS, they were subsequently excluded for 45 seconds. The exclusion window was 1 m/z below, and 1.6 m/z above the exclusion mass.

Polypeptide Identification from MS/MS Data

Database searching was performed using the SWIFT workflow tool developed in-house. SWIFT coordinates the generation of database search files (using extract_msn software from ThermoFisher Scientific), initiates database searches using MASCOT, Sequest, and X!Tandem search engines, and integrates these search results using Scaffold (Ver. 1_07_00, Proteome Software, Portland, Oreg.). Database searches were done against a subset of the SwissProt database (January 2007) obtained using the Bioworks 3.2 (Thermo Fisher) database utility to select human, bovine, and vaccinia proteins. Bovine proteins were included in the database since cell cultures were supplemented with fetal calf serum. Database searches were performed with a precursor mass tolerance of 7 parts-per-million (ppm), fragment ion mass tolerance of 0.6 mass units, and without any protease specificity. Single oxidation on methionine residues was considered as a variable modification. The database was appended with decoy protein entries consisting of randomized protein sequences (MASCOT utility) for estimating the false-positive rate resulting in a database of 43,400 entries (including the randomized decoy entries). Results from all analyses of all SCX fractions were combined by Scaffold and exported to an Excel spreadsheet.

The Scaffold program (Proteome Software) was used to combine search results from all of the LC-MS/MS analyses and to calculate polypeptide identification probabilities using Scaffold's implementation of PolypeptideProphet (Keller et al., Anal. Chem., 74(20):5383-5392 (2002) and Nesvizhskii et al., Anal. Chem., 75(17): 4646-4658 (2003)). An export function within Scaffold (Spectrum Report) was used to create a text file of all search results passing a lenient filter threshold of 80% protein probability, with at least 1 polypeptide identified above an 80% polypeptide probability threshold; 23,800 search results met those criteria, 1521 of these were from decoy polypeptides. Additional filtering was then applied to this dataset while estimating the false-positive rate (FPR) of identifications from the incidence of identifications from the decoy database using the formula: 2×# matches to decoy polypeptides/(number of true positives+number of false positives) as described elsewhere (Elias et al., Nat. Methods, 4(3):207-214 (2007)). The FPR was calculated as a function of thresholds for the following scoring parameters: Sequest cross-correlation score (XCorr), the difference between the top two normalized cross-correlation scores (ΔC_(n)), Mascot Ion Score, and mass error of the precursor mass.

Results

HLA Class I Polypeptide Identification by Tandem Mass Spectrometry

FIG. 1 provides an overview of the protocol used to sequence HLA class I polypeptides isolated from B-cells after infection with vaccinia. Sixteen strong cation exchange fractions were analyzed by nano-scale liquid chromatography coupled with tandem mass spectrometry (nLC-MS/MS) on the LTQ-Orbitrap. Two data sets were acquired, each using multiple injections as described above. The initial data set was acquired with external mass calibration, followed by a second data set collected with internal mass calibration using lock masses at m/z 391.2843 and m/z 445.1200 as described elsewhere (Olsen et al., Mol. Cell Proteomics, 4(12):2010-2021 (2005)). These analyses generated 214,800 MS/MS spectra that were searched against the human, bovine, and Vaccinia subset of proteins in the SwissProt database (January 2007 version).

Polypeptide sequencing by mass spectrometry involves matching MS/MS fragmentation spectra against theoretical fragmentation spectra calculated for any polypeptide sequence in the database within a tolerance window of the molecular weight of the polypeptide as measured by the mass spectrometer. As a result, every database search will return a result, and each of these matches to a sequence must be evaluated for their validity. A variety of scoring metrics exist from which a threshold is established for accepting or rejecting the search result from any MS/MS spectrum. The goal of that threshold is to minimize the number of incorrect or random matches that are accepted (false positives) while also trying to minimize the rejection of correct sequence matches (false negatives). Most of these scoring criteria have been developed within the context of identifying polypeptides generated from cleavage of proteins with trypsin. Trypsin cleaves on the C-terminal side of arginine and lysine and this cleavage specificity greatly reduces the list of candidate polypeptide sequences that are matched against the experimental spectra. The basic C-terminus of tryptic polypeptides provided by the Arg and Lys side chains, favorably directs fragmentation during MS/MS, influencing the scores from the database search results.

HLA class I polypeptides are not constrained to a basic C-terminal amino acid. For most common alleles, hydrophobic amino acids predominate in the C-terminal position, though basic residues such as Lys, Arg, Pro, or His, are often found somewhere in the C-terminal half of the polypeptide. Also not requiring Lys or Arg as the C-terminal amino acid from the database, greatly increases the number of candidate sequences whose theoretical fragmentation spectrum must be matched against the experimental fragmentation spectrum. Because of these differences we implemented the use of decoy database entries during the search as described elsewhere (Elias et al., Nat. Methods, 4(3):207-214 (2007)). For each protein in the database an additional entry is created with its amino acid sequence randomized, and labeled as such, in its accession identifier. During the database search these decoy proteins compete against authentic proteins for the best match to experimental spectra. Search results that identify polypeptides from a decoy protein are known to be incorrect. The rate of matches to polypeptides from the decoy proteins is used to estimate the false-positive rate as described above (Polypeptide identification from MS/MS data).

Search results are summarized in Table 1 at the estimated 1% and 5% FPR. 5915 MS/MS spectra were identified at the 1% FPR (30 matches against decoy polypeptides), representing 2731 unique sequences. 65 of these polypeptides were unique to vaccinia virus, originating from 44 vaccinia virus proteins. At the 5% FPR, the number of matched spectra increased from 5915 to 12,819 (313 matches against decoy polypeptides), 5601 of which were unique sequences. Of these 5601 unique sequences, 116 polypeptides originated from 61 vaccinia proteins.

TABLE 1 Summary of polypeptides identified by two-dimensional liquid chromatography and tandem mass spectrometry. 1% FPR^(a) 5% FPR^(a) # MS/MS spectra identified^(b) 5915 12819 # Unique sequences^(c) 2731 5601 # Unique vaccinia sequences^(d) 65 116 # Vaccinia proteins represented^(e) 44 61 ^(a)Database search results summarized at the 1% false positive rate (FPR) and the 5% FPR. ^(b)Number of tandem mass spectrometry (MS/MS) search results surpassing scoring thresholds that characterize the 1% and 5% FPR. Includes results for polypeptides identified in multiple strong cation exchange fractions, multiple MS/MS spectra from the same precursor m/z, at multiple charge states, and from multiple database entries containing the identified sequence. ^(c)Number of unique polypeptide sequences identified, from all species represented in the database (human, bovine, vaccinia proteins). ^(d)Number of polypeptide sequences identified that are unique to vaccinia proteins. ^(e)Number of vaccinia proteins represented by the identified vaccinia polypeptides.

The naturally processed and presented vaccinia polypeptides identified were listed in Table 2 and Table 3. The polypeptides were sorted by the open reading frame (ORF) they originated from and were marked whether they were identified within database search scoring criteria that characterized the 5% FPR (italicized) or the 1% FPR (non-italicized). Vaccinia polypeptide sequences identified at the 5% or better FPR were selected for additional studies to characterize their immunogenic properties.

TABLE 2 Class I polypeptides from Vaccinia virus identified by two-dimensional liquid chromatography and tandem mass spectrometry after Vaccinia infection of human B-cells. SEQ CBS ID Polypeptide Vaccinia Putative IC50 NO: sequence^(a) ORF strain^(b) Other pox viruses^(c) allele^(d) BIMAS^(e) (nM)^(f) SYFPEITHI^(g)  1 ILIRGIINV A C, V None A*0201 ORF T  2 AQITTDDLVKSY A10L C, V Vr, Cm, Cw, Mn, Ra B*1501  3 VQAVTNAGKIVY A12L A, C, V Vr,  Cm,  Cw,  Mn, B*1501 Ra  4 S ^(ox) MADVSIKTNSV A1L A, C, V Cm, Cw A*0201  5 LLFEDIIQNEY A23R A, C, T, V Vr, Cm, Cw, Mn B*1501 114  6 FTVNIFKEV A24R A, C, T, V Vr, Cm, Cw, Mn, Ra A*0201 8.4 195 16  7 GDKFTTRTSQKGTVAY A24R A, C, T, V Vr, Cm, Cw, Mn, Ra B*1501  8 ILYDPETDKPY A24R A, C, T, V Vr, Cm, Cw, Fw, Mn, B*1501 81 Ra  9 TTRTSQKGTVAY A24R A, C, T, V Vr, Cm, Cw, Mn, Ra B*1501 10 VIINSTSIF A24R A, C, T, V Vr, Cm, Cw, Mn, Ra B*1501 10.0 223 13 11 LTREMGFLVY A29L A, C, T, V Vr, Cm, Cw, Mn, Ra B*1501 17.4 132 15 12 TVINEDIVSKLTF A29L A, C, T, V Vr, Cm, Cw, Mn, Ra B*1501 13 TLRFLEKTSF A31R C, V Vr, Cm, Cw, Mn B*1501 72.0 507 11 14 VQIDVRDIKY A35R C, V Cm, Cw B*1501 52.8 131 23 15 YIIGNIKTV A35R C, V Cm, CwRa A*0201 101.2 143 29 16 IQYPGSEIKGNAY A44L A, V Cm, Cw B*1501 17 IQYPGSKIKGNAY A44L C Ra B*1501 18 IQYPGSKIKGNAYF A44L C Ra B*1501 19 KISNTTFEV A44L A, C, V Cm, Cw, Mn, Ra A*0201 194.1 10 23 20 LLISADDVQEIRV A44L A, C, V Vr, Cm, Cw, Mn, Ra A*0201 21 TLYDISPGHVYA A44L A, C, V Cm, Cw, Mn, Ra NA 22 YPGSKIKGNAY A44L C Ra B*1501 8.183 23 FQQKVLQEY A46R C, V Vr, Cm, Cw, Ra B*1501 476 24 FQQKVLQEY A48R A, C, T, V Vr, Cm, Cw, Mn, Ra B*1501 160.0 112 21 25 VAYAAAKGASM A48R A, C, T, V Cm, Cw, Ra NA 14.028 26 I^(ox)MNNPDFKTTY^(h) A49R C, V Vr, Cw B*1501 97 27 VQKQDIVKLTVY A52R C, V Cw B*1501 28 KLFNEDLSSKY A7L A, C, T, V Vr, Cw, Mn B*1501 183 29 LIQEIVHEV A7L A, C, T, V Vr, Cm, Cw, Mn A*0201 153.3 18 29 30 LVIENDSQF A8R A, C, V Vr, Cm, Cw B*1501 1.1 215 19 31 KLYKSGNSHIDY B12R A, C, V Cw, Ra B*1501 32 RVFAPKDTWSVF B12R A, C, V Cw, Ra B*1501 33 KVSAQNISF B13R C, V Vr, Cm, Cw, Mn, Ra B*1501 2.4 117  7 34 GQLYSTLLSF B15R C, V Vr, Cm, Ra B*1501 96.0 106 21 35 LQYAPRELLQY B1R A, C, V Vr, Cm, Cw, Mn B*1501 78 36 HCYL ^(ox) MNEGFES B21R, C Cw NA 39.041 C15L 37

C11R A, T None A*0201 83.5 4 27 38 KIKDDFQTVNF C12L C, V Vr, Cm, Cw, Mn B*1501 731 39 KIYGSDSIEF C12L C, V Vr, CwMn B*1501 14.4 229 12 40 YV ^(ox) MGGVYTTY C2L C, T, V Cm, Cw, Ra B*1501 6.3 68 23 41 KLSDSKITV D13L A, C, T, V Vr, Cm, Cw, Mn A*0201 998.1 21 24 42 VLSLELPEV D13L A, C, T, V Vr, Cm, Cw, Mn A*0201 271.9 14 28 43 ILVPNINILKI D6R A, C, T, V Vr, Cm, Cw, Mn NA 78 44

D6R A, C, T, V Vr, Cm, Cw, Fw, Mn, A*0201 46 Ra 45 RLKPLDIHY D8L A, C, T, V Cm, Cw, Ra B*1501 172.8 135 23 46 YAIDVSKVKPL E10R C Vr, Cm, Cw, Mn A*0201 1.337 47 GKASQNPSK^(ox)MVY E5R C, D Cw, Ra B*1501 48 IGKASQNSPK ^(ox) MVY E5R C, D Cw, Ra B*1501 49 KLFSDISAI E5R C, D, V Vr, Cm, Cw, Ra NA 310.7 8 25 50 SQNPSK ^(ox) MVY E5R C, D Cw, Ra B*1501 52.8 88 22 51 LARLGLVL E6R C, V Vr, Cm, Cw, Mn, Ra A*0201 52 GSFSGRYVSY E8R C, V Vr, Cm, Cw, Mn, Ra B*1501 1.2 204 15 53 KQKFPYEGGKVF E9L A, C, V Vr, Cm, Cw, Ra B*1501 54 IQHRQQLELAY F11L C, P Ra B*1501 83 55 IQKDINITH F11L C, P Vr, Cm, Cw, Ra NA 0.0 42.108  4 56 MLTEFLHYC F11L C, P Vr, Cw, Mn, Ra NA 1,664.5 105 17 57 LF^(ox)MDEIDHESY^(h) F12L C, P Vr, Cm, Cw, Mn, Ra B*1501 566 58 VQILMKTANNY F12L C Vr, Cm B*1501 106 59 KQISISTGVLY F16L C Vr, Cm, Cw, Mn B*1501 85 60 RVKQISISTGVLY F16L C Vr, Cm, Cw, Mn B*1501 61 IL ^(ox) MDNKGLGVRL F1L A, C, P, T,  Cw A*0201 V 62 RQLPTKTRSY F1L A, C, P, T,  Vr, Cm, Cw, Mn, Ra B*1501 96.0 80 25 V 63 ILKSEIEKATY G4L C, V Cw B*1501 374 64 ILIEIIPKI H4L A, C, V Vr, Cm, Cw, Mn, Ra NA 167.2 4 31 65 ITNKADTSSF H5R C, V Vr B*1501 2.6 276 10 66 IIKEDISEY H7R A, C, T, V Vr, Cm, Cw, Mn, Ra B*1501 42.9 171 19 67 YSKKFQESF IlL A, C, P, T,  Cm, Cw, Mn B*1501 6.0 185 11 V 68 KLLLGELFFL J3R A, C, V Vr, Cm, Cw, Mn A*0201 20,297.3 7 27 69 KQKGHNKFPVNF J4R C, V Vr, Cm, Cw, Mn B*1501 70 VVIGNTLIKY J6R A, C, V Vr, Cw, Mn, Ra B*1501 2.9 242 22 71 K ^(ox) MIIEKHVEY K1L C, V None B*1501 2.4 103 16 72 ^(ox) MIIEKHVEY K1L C, V None B*1501 13.2 154 18 73 SLLFIPDIKL K1L C, V Vr, Cw, Mn, Ra A*0201 79.0 61 25 74 SQFDDKGNTALY K1L C, V Cm, CwMn, Ra B*1501 75 VLLDDAEIAK^(ox)M K1L V Cm, Cw, Mn, Ra NA 55 76 VLLDDAEIAK^(ox)MII^(h) K1L V Cm, Cw, Mn, Ra NA 77 KLVGKTVKV K3L C, V Cm, Cw, Mn A*0201 243.3 54 30 78 ITYPKALVF K6L C, V Cw, Mn B*1501 4.1 168 11 79 MMIDDFGTARGNY K6L C, V None B*1501 80 L ^(ox) MKFDDVAIRY K7R A, C, V Cw B*1501 147 81 RLYKEL^(ox)MKF^(h) K7R A, C, V Cm, Cw, Ra B*1501 40.0 763 20 82 HIIKEFMTY N2L C, V Vr, Cm, Cw, Mn, Ra B*1501 11.0 797 18 83 SIIAILDRF N2L V Vr, Cm, Cw, Ra B*1501 20.0 3.775 16 ^(a)Non-italicized polypeptides were identified at a 1% false positive identification rate (1% FPR); italicized polypeptides were identified within scoring criteria characteristic of a 5% FPR. Polypeptide sequences in boldface are predicted to be strong binders (IC₅₀ < 50 nM) by the epitope prediction algorithm at: http://www.cbs.dtu.dk/services/NetMHC/ (accessed Jan. 4, 2008). ^(ox)M denotes that the polypeptide contains an oxidized methionine (not considered incalculation of IC₅₀ values). ^(b)Epitope is common to the following vaccinia strains: A, Ankara; C, Copenhagen; D, Dairen I; P, L-IVP; T, Tian Tan; V, Western Reserve as listed in the SwissProt database. ^(c)Epitope also found in the following common poxviruses: Vr, variola; Cm, camelpox; Cw, cowpox; Fw, fowlpox; Mn, monkeypox, Ra, rabbitpox; None, not found in other poxviruses as reported in the NCBI nr database (http://www.ncbi.nlm.nih.gov/). ^(d)Putative association of polypeptide with A*0201 or B*1501 alleles based upon the C-terminal amino acid: amino acids V and L classified as A2; amino acids F and Y classified as B15; NA, not assigned. ^(e)Score is proportional to predicted affinity. Bioinformatics and Molecular Analysis Section, National Institute of Health, http://www-bimas.cit.nih.gov/molbio/hla bind/ ^(f)Center for Biological Sequence Analysis, Technical University ofDenmark, http://www.cbs.dtu.dk/services/NetMHC/ ^(g)Score is proportional to predicted affinity. Department of Immunology, University of Tübingen, http://www.syfpeithi.de/ ^(h)Polypeptide was identified withoxidized and non-oxidized forms of methionine ^(i)Polypeptide was identified in four oxidative forms: without methionine oxidation, oxidized at the N- terminal methionine, oxidized at the position-7 methionine, and oxidized at both methionines.

TABLE 3 Class I polypeptides from vaccinia virus identified by two-dimensional liquid chromatography and tandem mass spectrometry after vaccinia infection of human B-cells. SEQ CBS ID Polypeptide Vaccinia Putative IC50 NO: sequence^(a) ORF strain^(b) Other pox viruses^(c) allele^(d) BIMAS^(e) (nM)^(f) SYFPEITHI^(g)  84 GLLDRLYDL O1L C, V Cm, Cw, Ra A*0201 745.4 10 29  85 IVIEAIHTV A48R A, C, T, V Vr, Cm, Cw, Mn, Ra A*0201 97.6 53 27  86 ILSDENYLL A6L C, V Vr, Cm, Cw, Mn, Ra A*0201 148.9 10 24  87 ILDDNLYKV G5R C Vr, Cm, Cw, Mn, Ra A*0201 446.0 4 30  88 KLFTHDIML D12L A, C, V Vr, Cm, Mn A*0201 276.6 15 22  89 KIDYYIPYV E2L C, V Vr, Cm, Cw, Mn, Ra A*0201 169.4 2 24  90 FLTSVINRV F12L C Vr, Cm, Cw, Mn, Ra A*0201 735.9 5 25  91 NLFDIPLLTV F12L C, P Vr, Cm, Cw, Mn, Ra A*0201 2,426.7 7 29  92 ^(ox) MQKFTILEY ^(h) A, C, V Vr, Cm, Cw, Mn B*1501 288.0 65 23  93 SQIFNIISY A17L C, V Vr, Cm, Cw, Mn B*1501 96.0 74  9  94 ALDEKLFLI A23R A, C, T, V Vr, Cm, Cw, Mn NA 228.2 6 27  95 HMIDKLFYV A23R A, C, T, V Vr, Cm, Cw, Mn A*0201 513.8 2 26  96 QIDVRDIKY A35R C, V Cm, Cw B*1501 1.8 12.810 16  97 SIMDFIGPYI A35R C, V Cm, Cw, Mn, Ra NA 119.7 11 21  98 YAAAKGASM A48R A, C, T, V Cm, Cw, Ra NA 0.3 4.895 17  99 ILQNRLVYV A52R C, V Cw A*0201 1,495.7 13 28 100 IQFMHEQGY B1R A, C, V Vr, Cm, Cw, Mn B*1501 52.0 106 21 101 TLLDHIRTA B22R, C Cw, Ra NA 34.7 84 23 C16L 102 RQFYNANVL C2L C, T, V Cm, Cw, Ra A*0201 6.960 11 103 ILKINSVKY D12L A, C, V Vr, Cm, Mn B*1501 187.2 309 22 104 LLLETKTILV E9L A, C, V Vr, Cm, Cw, Mn, Ra A*0201 1,793.7 11 26 105 SLSNLDFRL F11L C, P Cw, Mn, Ra A*0201 123.9 30 23 106

F1L A, C, P,T,  Cw A*0201 1,793.7 8 26 V 107 QLIYQRIYY F2L C, P,V Vr, Cm, Cw, Mn, Ra B*1501 26.4 250 21 108 SLKDVLVSV G5.5E A, C, T, V Vr, Mn A*0201 23.0 18 30 109 VPLPCQL ^(ox) MY G5R C Vr, Cm, Cw, Mn, Ra B*1501 2.8 18.814 12 110 NTIDKSSPL I1L A, C, T, V Mn A*0201 1.2 3.795 18 111 TQFNFNGHTY I1L A, C, P,T,  Cm, Cw, Mn B*1501 88.0 70 22 V 112

J6R A, C, V Vr, Cm, Cw, Mn, Ra B*1501 2.6 49 11 113 YLFDYPHFEA K3L V None NA 2,010.7 8 20 114 IINDKGKQY M1L C, V Vr, Cw, Mn, Ra B*1501 14.3 423 18 115 QAIEPSGNNY M1L C, V Cw, Ra B*1501 2.2 146 12 116 ILFRMIETY N1L C, V Vr, Cw B*1501 57.2 208 24 ^(a)Non-italicized polypeptides were identified at a 1% false positive identification rate (1% FPR); italicized polypeptides were identified within scoring criteria characteristic of a 5% FPR. Polypeptide sequences in boldface are predicted to be strong binders (IC₅₀ < 50 nM) by the epitope prediction algorithm at: http://www.cbs.dtu.dk/services/NetMHC/ (accessed Jan. 4, 2008). ^(ox)M denotes that the polypeptide contains an oxidized methionine (not considered incalculation of IC₅₀ values). ^(b)Epitope is common to the following vaccinia strains: A, Ankara; C, Copenhagen; D, Dairen I; P, L-IVP; T, Tian Tan; V, Western Reserve as listed in the SwissProt database. ^(c)Epitope also found in the following common poxviruses: Vr, variola; Cm, camelpox; Cw, cowpox; Fw, fowlpox; Mn, monkeypox, Ra, rabbitpox; None, not found in other poxviruses as reported in the NCBI nr database (http://www.ncbi.nlm.nih.gov/). ^(d)Putative association of polypeptide with A*0201 or B*1501 alleles based upon the C-terminal amino acid: amino acids V and L classified as A2; amino acids F and Y classified as B15; NA, not assigned. ^(e)Score is proportional to predicted affinity. Bioinformatics and Molecular Analysis Section, National Institute of Health, http://www-bimas.cit.nih.gov/molbio/hla bind/ ^(f)Center for Biological Sequence Analysis, Technical University ofDenmark, http://www.cbs.dtu.dk/services/NetMHC/ ^(g)Score is proportional to predicted affinity. Department of Immunology, University of Tübingen, http://www.syfpeithi.de/ ^(h)Polypeptide was identified withoxidized and non-oxidized forms of methionine ^(i)Polypeptide was identified in four oxidative forms: without methionine oxidation, oxidized at the N- terminal methionine, oxidized at the position-7 methionine, and oxidized at both methionines.

FIG. 2 shows the distribution of polypeptide lengths and putative allele from the Vaccinia subset (FIG. 2A) as well as all class I polypeptides identified at the 5% FPR rate (FIG. 2B). The majority of the polypeptides were 9-mers in both the full set of polypeptides as well as the Vaccinia subset. However, there were a significant number of polypeptides longer than 11 amino acids in the full set of identified polypeptides as well as in the Vaccinia subset. Since the W6/32 antibody used to isolate the HLA-polypeptide complexes had affinity for each of the major HLA class I alleles, the list of identified polypeptides reflected the allotypes of the host cell line; in this case HLA-A*0201, B*1501, and C*03. As a first approximation, identified polypeptides were associated with an allele by comparing the C-terminal amino acid to the reported binding motifs at the P9 position (Rammensee et al., Immunogenetics, 41:178-228 (1995)). Polypeptides terminating in L or V were assigned to A*0201, and polypeptides terminating in F or Y were assigned to B*1501, while the remaining polypeptides were designated as “not assigned.” FIGS. 2C and 2D shows an estimate of the distribution of polypeptides among the A and B alleles, and while the method of associating a polypeptide with its putative allele was rudimentary, it clearly showed a significant subset of the polypeptides being presented by the B allele. There was also an unknown contribution of polypeptides from the HLA-C allele, although the density of HLA-C molecules on the surface of cells has been reported as being 6-fold less than that of the A and B alleles (Snary et al., Eur. J. Immunol., 7(8):580-585 (1977) and Neisig et al., J. Immunol., 160(1):171-179 (1998)).

The vaccinia polypeptides identified also beared evidence of the genetic heterogeneity of the Dryvax strain (Osborne et al., Vaccine, 25(52):8807-8832 (2007)). For example, two forms of the same polypeptide from ORF A44L were identified: IQYPGSKIKGNAY (SEQ ID NO:17) from the Copenhagen strain, and IQYPGSEIKGNAY (SEQ ID NO:16) from the Western Reserve and Ankara strains of vaccinia. At first glance, these two sequences were flagged as redundant identities of the same polypeptide, since they varied only in one amino acid residue resulting in a mass difference of one Dalton. However, these sequences were assigned from two distinctly different polypeptides with IQYPGSEIKGNAY (SEQ ID NO:16) identified from SCX fractions 4 and 5, and IQYPGSKIKGNAY (SEQ ID NO:17) identified from SCX fractions 14-16. FIGS. 3A and 3B shows the annotated MS/MS spectra for the two polypeptides. Orbitrap spectra for each precursor mass (inset) illustrated the difference in mass for the two polypeptides (0.5 on the m/z axis, where z=2 as shown by the isotope spacing). Fragment ions were observed from the N-terminal end of the polypeptide (b-ions, highlighted in dark gray) and from the C-terminal end of the polypeptide (y-ions, highlighted in light gray), while the y- and b-ions highlighted within the dashed box delineated the sequence difference between the two polypeptides. Table 2 and Table 3 contains other instances where polypeptides were identified that were unique to specific Vaccinia strains.

Comparison of Directly Identified Vaccinia Class I Polypeptides to Predictive Algorithms

The Vaccinia protein sequences represented by this set of polypeptides were submitted to three algorithms for predicting potential epitopes according to their predicted alleles. Results from the NetMHC (Buus et al., Tissue Antigens, 62(5):378-384 (2003) and Nielsen et al., Protein Sci., 12(5):1007-1017 (2003)), and BIMAS (Parker et al., J. Immunol., 152:163-175 (1994)) and SYFPEITHI (Rammensee et al., Immunogenetics, 50:213-219 (1999)) algorithms are shown in Table 3. The NetMHC algorithm used a neural network approach to determine likely epitopes from protein sequence, calculated a predicted binding affinity to HLA molecules, and classified this binding as being strong (IC₅₀<50 nM), weak (IC₅₀>50 nM and <500 nM), or less than weak (IC₅₀>500 nM) (http://www.cbs.dtu.dk/services/NetMHC/, accessed Jan. 4, 2008). FIG. 4 summarizes how the polypeptides that were directly identified by MS were predicted by the NetMHC algorithm to bind with HLA molecules. Twenty-seven (22%) of the Vaccinia polypeptides identified by MS were predicted by the algorithm to be strong binders with another 49 (41%) predicted to be weak binding. Twenty-four polypeptides identified by MS/MS were not predicted by the algorithm, 23 because of excess length.

Additionally, a comparison was made of the list of identified Vaccinia polypeptides to Vaccinia epitopes, from all alleles, contained in the Immune Epitope Database and Analysis Resource (IEDB, http://www.immuneepitope.org/home.do, accessed Jan. 4, 2008) (Peters et al., Nat. Rev. Immunol., 7(6):485-490 (2007)). From the list of 116 directly identified polypeptides, 23 were an exact match to an IEDB database record, while another 7 polypeptides were contained within a Vaccinia epitope from the IEDB database. These results demonstrated both the complementary information provided by direct identification of epitopes by MS, and the limitations inherent in relying solely on computer algorithms for understanding the spectrum of polypeptides presented by natural infection.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. 

What is claimed is:
 1. A kit for vaccinia epitope specific quantitation of CD8+ T cells, wherein said kit comprises an MHC class I tetramer for performing flow cytometry comprising a polypeptide selected from the group consisting of SEQ ID NOs: 1-82 and
 83. 